Difunctional glutathione synthetase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a bifunctional glutathione synthetase mutant and application thereof. The amino acid sequence of the bifunctional glutathione synthetase mutant is shown as SEQ ID NO.3. The enzyme mutant can efficiently catalyze L-glutamic acid, L-cysteine and glycine to generate glutathione, the concentration reaches 38.3 g / L, and the enzyme activity is improved by 4.2 times compared with that before mutation. The glutathione synthesized by using the bifunctional glutathione synthetase mutant provided by the invention has the advantages of high enzyme activity, short conversion time, simple and convenient process, low production cost and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a bifunctional glutathione synthetase mutant and its application. BACKGROUND

[0002] Glutathione is a kind of non-protein thiol compound condensed by L-glutamic acid, L-cysteine and glycine, which widely exists in animals, plants and microorganisms. Reduced glutathione has important functions in living tissues, such as antioxidant stress, regulation of cellular redox homeostasis, enhancement of host immunity, etc., and is widely used in food, medicine, cosmetics and other fields, such as food preservation, whitening and freckle removal, and reduction of pathological damage.

[0003] At present, there are many methods for preparing glutathione, such as solvent extraction, chemical synthesis, fermentation and enzyme method.

[0004] Solvent extraction method is an early method for producing glutathione, which is based on the difference in solubility of glutathione in different solvents. Glutathione is extracted from biological tissues with high glutathione content by extraction, enzyme treatment, separation and purification. This method has low production yield, high cost and serious pollution due to complicated process flow, so its application in industry is facing great challenges.

[0005] Chemical synthesis method is the earliest method for industrial production of glutathione, and the production process is relatively mature. Three precursor amino acids glutamic acid, L-cysteine and glycine are synthesized into glutathione through three chemical reactions of group protection, condensation and deprotection. This method has many defects, such as long reaction time, many reaction steps, difficult operation and difficult separation, etc. Therefore, the glutathione produced by this method has low purity and unstable biological titer, which restricts its application scenarios.

[0006] Fermentation method is a common method for industrial production of glutathione. The gene encoding glutathione synthetase or bifunctional glutathione synthetase is cloned into bacteria or yeast to obtain glutathione by fermentation. Patents CN201810844388 and CN201680013630 express exogenous bifunctional glutathione synthetase gene in yeast and E. coli respectively, to achieve the purpose of fermentation production of glutathione, but the yield is low and the downstream process is complex, which is not suitable for mass production.

[0007] Enzymatic synthesis of glutathione has the advantages of fast reaction speed, mild conditions, high substrate concentration, easy separation and purification, and gradually becomes the most potential method for synthesizing glutathione. Patents CN 200910199345.3 and CN201210201691.2 use bifunctional glutathione synthetase to synthesize glutathione in one step. However, the enzyme activity and catalytic reaction concentration of the existing glutathione synthetase still need to be improved. With the continuous expansion of the application scenarios of glutathione, the market demand for glutathione is also increasing. Therefore, it is imperative to develop a high-enzyme-activity glutathione synthetase and provide an efficient, simple and low-cost method for preparing glutathione. SUMMARY

[0008] The present application aims to overcome the deficiencies in the prior art and provide a bifunctional glutathione synthetase mutant and its application.

[0009] In order to achieve the purpose of the present application, the following technical solutions will be adopted.

[0010] A bifunctional glutathione synthetase mutant and its application, characterized in that the bifunctional glutathione synthetase mutant is obtained by mutating one or more amino acid residue sites in the bifunctional glutathione synthetase represented by the amino acid sequence SEQ ID NO. 1, wherein the mutated amino acid residue sites are the 163rd glycine mutated to serine, the 209th alanine mutated to serine, the 259th serine mutated to threonine, the 396th aspartic acid mutated to valine, and the 604th glutamic acid mutated to glutamine, and the amino acid sequence of the bifunctional glutathione synthetase mutant is shown in SEQ ID NO. 3; the bifunctional glutathione synthetase mutant can efficiently catalyze L-glutamic acid, L-cysteine and glycine to generate glutathione.

[0011] As a preferred embodiment of the present application, the bifunctional glutathione synthetase is derived from Listeria Listeria monocytogenes , and the gene sequence is shown in SEQ ID NO. 2.

[0012] As a preferred embodiment of the present application, the method for catalyzing the synthesis of glutathione by the bifunctional glutathione synthetase mutant is to use a buffer containing 10.0-20.0 g / L of L-glutamic acid, 8.0-17.0 g / L of L-cysteine, 5.0-11.0 g / L of glycine and 35.0-70.0 g / L of adenosine triphosphate as the reaction system, add genetically engineered bacterial cells or their crude enzyme solution with bifunctional glutathione synthetase mutant activity, react at pH 7-10 and 25-45℃, and determine the content of glutathione by HPLC.

[0013] As a preferred embodiment of the present invention, the bifunctional glutathione synthase mutant genetically engineered bacteria is obtained by heat shock transformation of Escherichia coli BL21(DE3) competent cells into expression vector pET-28a carrying the bifunctional glutathione synthase mutant gene.

[0014] As a preferred embodiment of the present invention, the crude enzyme solution is obtained by ultrasonic disruption or high-pressure homogenization of whole cells of genetically engineered bacteria capable of expressing mutants.

[0015] As a preferred embodiment of the present invention, the bifunctional glutathione synthase mutant gene can encode a bifunctional glutathione synthase mutant.

[0016] Compared with the prior art, the beneficial effect of the present invention is that it provides a high-enzyme-activity bifunctional glutathione synthase mutant and a method for efficient, simple and low-cost synthesis of glutathione.

[0017] (1) This invention provides a high-enzyme-activity bifunctional glutathione synthase mutant. Specifically, the amino acid sequence of the bifunctional glutathione synthase has undergone a specific mutation, which improves the enzyme's catalytic activity.

[0018] (2) This invention provides an enzymatic method for synthesizing glutathione. A bifunctional glutathione synthase mutant is used to efficiently catalyze the synthesis of glutathione from L-glutamic acid, L-cysteine, and glycine, offering advantages of being green, environmentally friendly, mild, and highly efficient. Attached Figure Description

[0019] Figure 1 Bifunctional glutathione synthase ( Lm A roadmap for the synthesis of glutathione by GshF mutants.

[0020] Figure 2 : Lm Electrophoresis diagram of GshF mutant recombinant plasmid nucleic acid.

[0021] Figure 3 : Lm Fermentation induction curve of GshF mutant genetically engineered bacteria.

[0022] Figure 4 : Lm Electrophoresis diagram of GshF mutant protein expression. Detailed Implementation

[0023] The technical solutions of the present invention will now be clearly and completely described with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be operated according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0025] Table 1 shows the full Chinese names, three-letter abbreviations, and single-letter abbreviations of the 20 natural amino acids in this invention: Table 1. Full Chinese names, three-letter abbreviations, and single-letter abbreviations of 20 natural amino acids. Chinese name of amino acid Three-letter abbreviation One-letter abbreviation Arginine Arg R Histidine His H Lysine Lys K Aspartic acid Asp D Glutamic acid Glu E Serine Ser S Threonine Thr T Asparagine Asn N Glutamine Gln Q Cysteine Cys C Glycine Gly G Proline Pro P Alanine Ala A Isoleucine Ile I Valine Val V Leucine Leu L Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W In this patent, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, W85F indicates that amino acid W at position 85 is mutated to amino acid F, and so on.

[0026] The present invention relates to a method for altering the amino acid sequence of a bifunctional glutathione synthase (encoding gene with amino acid sequence as shown in SEQ ID NO. 1) by mutating the gene using whole-plasmid PCR amplification, based on the wild-type bifunctional glutathione synthase gene shown in SEQ ID NO. 2. This alters the enzyme's amino acid sequence, thereby changing its structure and function. A bifunctional glutathione synthase mutant is then obtained through targeted screening.

[0027] Specifically, the preparation method includes the following steps: (1) preparing a bifunctional glutathione synthase ( Lm (1) Genes of the corresponding mutation sites of GshF were constructed into the pET-28a expression vector to obtain recombinant plasmids carrying the target enzyme gene; (2) The recombinant plasmids were transformed into host bacterial cells, preferably Escherichia coli BL21(DE3), to obtain the corresponding engineered strains; (3) The engineered strains were inoculated into LB resistant medium and cultured, and then cultured at a certain ratio. After a certain period of culture, isopropyl-β-D-galactoside (IPTG) or lactose was added to induce culture for a certain period of time. The bacterial cells were collected by centrifugation and subjected to high pressure disruption; (4) The enzyme activity of the bifunctional glutathione synthase mutant was determined.

[0028] Example 1: Bifunctional glutathione synthase (Lm Construction of GshF mutant library This embodiment relates to a bifunctional glutathione synthase ( Lm The discovery and mutation process of GshF includes the following steps: (1) Select template sequence The amino acid sequences of bifunctional glutathione synthases reported in existing literature were retrieved, and their species origin, catalytic activity data (substrate profile, activity, enzymatic properties, etc.), protein structure information, key amino acids in the protein structure, and catalytic mechanisms were compiled. Finally, a glutathione synthase derived from Listeria was selected. Listeria monocytogenes The GshF amino acid sequence (GenBank: SCU56489.1) is the template sequence.

[0029] (2) Lm Prediction of GshF mutation hotspots This invention obtained the three-dimensional structure prediction file of GshF from the AlphaFold protein structure prediction database. Through analysis of substrate channels, binding pockets and molecular docking structures, 10 key amino acid mutation sites were finally identified, including W85, G163, A209, S259, K318, D396, V478, E604, T717 and F748.

[0030] (3) Site-directed mutation The amino acids that play a key role in enzyme catalytic efficiency were identified, and mutant primers were designed using the degenerate codon NNK to target pET28a- GshF As a template for the source Listeria monocytogenes Site-directed mutagenesis was performed on the amino acid sequence of the bifunctional glutathione synthase of the strain. The mutation sites for library construction were W85, G163, A209, S259, K318, D396, V478, E604, T717, and F748. The beneficial mutation sites that increased enzyme activity were G163, A209, S259, D396, and E604.

[0031] Example 2: Induced expression of a bifunctional glutathione synthase mutant The constructed bifunctional glutathione synthase mutant was induced to express protein, and a crude enzyme solution was prepared. The specific steps included: Activation of strain: Add 50 μL of pET28a- GshF The mutant bacterial culture was inoculated into 5 mL of liquid LB medium containing 50 μg / mL kanamycin and activated in a shaker at 37℃ and 220 r / min for 12 h.

[0032] Cell collection: The activated bacterial culture was inoculated at a rate of 1% into 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37℃ and 220 r / min for 4 h in a shaker until the bacterial concentration OD was determined. 600 When the concentration of the inducing agent was 0.6–0.8, IPTG was added to a final concentration of 0.3 mmol / L, and expression was induced for 16 h in a shaker at 18 °C and 220 r / min. The cultured bacterial solution was centrifuged at 4000 r / min for 20 min, the supernatant was discarded, and the bacterial cell pellet was collected.

[0033] Preparation of crude enzyme solution: The collected bacterial cells were suspended in an appropriate amount of phosphate buffer (100 mmol / L, pH 7.2) and disrupted using an ultrasonic homogenizer. The cells were then centrifuged again to obtain intracellular supernatant and precipitate. The supernatant was the crude enzyme solution of the bifunctional glutathione synthase mutant.

[0034] Example 3 Enzyme activity detection of bifunctional glutathione synthase mutant Enzyme activity assay method: Lm The GshF mutant catalyzes the production of glutathione (GSH) from substrates L-glutamate, L-cysteine, and glycine, with ATP providing energy. The amount of GSH produced is then used to calculate... Lm Enzyme activity of GshF.

[0035] Enzyme activity unit (IU) definition: The amount of enzyme required to generate 1 μmol of glutathione per unit time under conditions of 37℃ and pH 8.0.

[0036] Lm The composition and concentration of the GshF enzyme activity assay reaction system are shown in Table 2: Table 2 Lm Composition of the GshF enzyme activity assay reaction system Reaction system Final concentration pH 8.0 Tris-HCl 500 mmol / L Glycine 120 mmol / L L-glutamic acid 100 mmol / L L-cysteine 120 mmol / L MgCl2 40 mmol / L ATP 100 mmol / L Enzyme 30 μg Water Supplemented to 500 μL Enzyme activity assay reaction steps: The above reaction system was reacted at 37℃ for 5 min, and then an equal volume of 2 mol / L hydrochloric acid was added to terminate the reaction. The GSH content was determined by centrifugation at 12000 r / min for 5 min.

[0037] GSH content detection method: using Wondasil C 18 The content of GSH was detected by an HPLC system with a column (4.6 × 250 mm, 5 μm) (Shimadzu, Kyoto, Japan) at 25 °C and 210 nm. The mobile phase was phosphate buffer (6.8 g of sodium dihydrogen phosphate and 2.2 g of sodium heptanesulfonate were dissolved in 1000 mL of water and the pH was adjusted to 3.0 with phosphoric acid) / methanol (96 / 4), with an injection volume of 10 μL.

[0038] The relative enzyme activity assay results of wild-type strain (WT) and mutants with different site combinations (M1-M6) are shown in Table 3: Table 3. Relative enzyme activities of wild-type strain (WT) and mutants with different site combinations (M1-M6) Name Mutant site Relative enzyme activity WT - 100.0% M1 G163S 165.1% M2 A209S 173.7% M3 G163S-A209S 247.4% M4 G163S-A209S-K318H 284.2% M5 G163S-A209S-S259T-D396V 334.6% M6 G163S-A209S-D396V-E604Q 420.7% As shown in Table 3, the enzyme activity of the mutant M6 (G163S-A209S-D396V-E604Q) obtained in this invention is significantly increased compared with that of the wild type, with an increase of 4.2 times.

[0039] Example 4: Synthesis of glutathione by a bifunctional glutathione synthase mutant A 1000 mL buffer reaction system (pH 8.0) containing 10.0 g / L L-glutamic acid, 8.5 g / L L-cysteine, 5.5 g / L glycine, and 35.0 g / L adenosine triphosphate was added to bring the concentration to 8.0 g / L. 10 g of genetically engineered bacterial cells with bifunctional glutathione synthase mutant activity were added. The reaction was carried out at 35 °C with stirring for 20 h. The glutathione content was determined by HPLC, and the GSH concentration was 20.8 g / L.

[0040] Example 5: Synthesis of glutathione by a bifunctional glutathione synthase mutant A 1000 mL buffer reaction system (pH 9.0) containing 15.0 g / L L-glutamic acid, 13 g / L L-cysteine, 8.0 g / L glycine, and 52.0 g / L adenosine triphosphate was added to bring the concentration to 10.0 g / L. 15 g of genetically engineered bacterial cells with bifunctional glutathione synthase mutant activity were added. The reaction was carried out at 45 °C with stirring for 24 h. The glutathione content was determined by HPLC, and the GSH concentration was 31.3 g / L.

[0041] Example 6: Synthesis of glutathione by a bifunctional glutathione synthase mutant A 1000 mL buffer reaction system (pH 10.0) containing 12.0 g / L L-glutamic acid, 10 g / L L-cysteine, 6.5 g / L glycine, and 42.0 g / L adenosine triphosphate was added to bring the concentration to 8.0 g / L. 10 g of genetically engineered bacterial cells with bifunctional glutathione synthase mutant activity were added. The reaction was carried out at 30 °C with stirring for 24 h. The glutathione content was determined by HPLC, and the GSH concentration was 24.9 g / L.

[0042] Example 7: Synthesis of glutathione by a bifunctional glutathione synthase mutant A 1000 mL buffer reaction system (pH 7.0) containing 10.0 g / L L-glutamic acid, 8.5 g / L L-cysteine, 5.5 g / L glycine, and 35.0 g / L adenosine triphosphate was added to bring the concentration to 8.0 g / L. 20 g of crude enzyme solution from genetically engineered bacteria with bifunctional glutathione synthase mutant activity was added. The reaction was carried out at 25°C with stirring for 22 h. The glutathione content was determined by HPLC, and the GSH concentration was 19.8 g / L.

[0043] Example 8: Synthesis of glutathione by a bifunctional glutathione synthase mutant A 1000 mL buffer reaction system (pH 8.5) containing 20.0 g / L L-glutamic acid, 17 g / L L-cysteine, 11.0 g / L glycine, and 70.0 g / L adenosine triphosphate was added to bring the concentration to 10.0 g / L. 20 g of genetically engineered bacterial cells with bifunctional glutathione synthase mutant activity were added. The reaction was carried out at 37°C with stirring for 24 h. The glutathione content was determined by HPLC, and the GSH concentration was 38.3 g / L.

[0044] The technical solutions of the present invention have been described in detail above with reference to the embodiments / drawings. However, the present invention is not limited to the above technical solutions. For those skilled in the art, after learning the contents described in the present invention, several equivalent changes and substitutions can be made without departing from the principle of the present invention. These equivalent changes and substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. A bifunctional glutathione synthetase mutant and use thereof, characterized in that, The bifunctional glutathione synthetase mutant is obtained by mutating one or more amino acid residue sites in the bifunctional glutathione synthetase shown in the amino acid sequence SEQ ID NO. 1, wherein the mutated amino acid residue sites are the 163rd glycine mutated into serine, the 209th alanine mutated into serine, the 259th serine mutated into threonine, the 396th aspartic acid mutated into valine, and the 604th glutamic acid mutated into glutamine, the amino acid sequence of the bifunctional glutathione synthetase mutant is shown in SEQ ID NO. 3; the bifunctional glutathione synthetase mutant can efficiently catalyze L-glutamic acid, L-cysteine and glycine to generate glutathione.

2. The bifunctional glutathione synthetase mutant according to claim 1, wherein, The bifunctional glutathione synthetase is derived from Listeria Listeria monocytogenes , the gene sequence of which is shown as SEQ ID NO.

2.

3. The bifunctional glutathione synthetase mutant according to claim 1, wherein, The method for synthesizing glutathione by using the bifunctional glutathione synthetase mutant is to use a buffer containing 10.0-20.0 g / L of L-glutamic acid, 8.0-17.0 g / L of L-cysteine, 5.0-11.0 g / L of glycine and 35.0-70.0 g / L of adenosine triphosphate as the reaction system, to add genetically engineered bacterial cells or crude enzyme liquid with the activity of the bifunctional glutathione synthetase mutant, to react at pH 7-10 and 25-45℃, and to determine the content of glutathione by using HPLC.

4. The bifunctional glutathione synthetase mutant according to claim 3, characterized in that, The bifunctional glutathione synthetase mutant gene engineering bacteria is obtained by transforming the expression vector pET-28a carrying the bifunctional glutathione synthetase mutant gene into Escherichia coli BL21(DE3) by heat shock method E. coli obtained in the BL21(DE3) competent cell.

5. The bifunctional glutathione synthetase mutant according to claim 3, wherein the mutant is a mutant of SEQ ID NO:

1. The crude enzyme liquid is obtained by ultrasonic or high-pressure homogenate disruption of whole cells of the genetically engineered bacteria capable of expressing the mutant.

6. The bifunctional glutathione synthetase mutant according to claim 4, wherein, The bifunctional glutathione synthetase mutant gene can encode the bifunctional glutathione synthetase mutant. The bifunctional glutathione synthetase mutant is obtained by mutating one or more amino acid residue sites in the bifunctional glutathione synthetase shown in the amino acid sequence SEQ ID NO. 1, wherein the mutated amino acid residue sites are the 163rd glycine mutated into serine, the 209th alanine mutated into serine, the 259th serine mutated into threonine, the 396th aspartic acid mutated into valine, and the 604th glutamic acid mutated into glutamine, the amino acid sequence of the bifunctional glutathione synthetase mutant is shown in SEQ ID NO. 3; the bifunctional glutathione synthetase mutant can efficiently catalyze L-glutamic acid, L-cysteine and glycine to generate glutathione. Listeria monocytogenes The method for synthesizing glutathione by using the bifunctional glutathione synthetase mutant is to use a buffer containing 10.0-20.0 g / L of L-glutamic acid, 8.0-17.0 g / L of L-cysteine, 5.0-11.0 g / L of glycine and 35.0-70.0 g / L of adenosine triphosphate as the reaction system, to add genetically engineered bacterial cells or crude enzyme liquid with the activity of the bifunctional glutathione synthetase mutant, to react at pH 7-10 and 25-45℃, and to determine the content of glutathione by using HPLC. E. coli The crude enzyme liquid is obtained by ultrasonic or high-pressure homogenate disruption of whole cells of the genetically engineered bacteria capable of expressing the mutant. The bifunctional glutathione synthetase mutant gene can encode the bifunctional glutathione synthetase mutant.

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